PROJECT SUMMARY/ABSTRACT The RNA exosome is an evolutionary-conserved 3'-5' riboexonuclease complex critically important for both precise processing and complete degradation of a variety of cellular RNAs. One of the most critical cellular functions of the exosome is the production of mature, properly trimmed rRNAs required within the ribosome. How the exosome plays a dual role in mediating both precise processing of some RNA targets and complete destruction of others including potentially damaging aberrant transcripts is not understood; however, one model is that a variety of associated co-factors dictate target specificity. Given, the crucial role of the RNA exosome in post-transcriptional regulation of RNA, it is not surprising that the complex is essential in the systems examined thus far including budding yeast. For this reason, the recent discovery that mutations in multiple genes that encode RNA exosome subunits, EXOSC2, EXOSC3, and EXOSC8, are linked to human disease was unexpected. Mutations in EXOSC3 cause Pontocerebellar Hypoplasia type 1b (PCH1b), which is an autosomal recessive neurodegenerative disease, while mutations in EXOSC2 cause a variety of distinct tissue-specific phenotypes including mild intellectual disability. The disease-causing mutations identified thus far are not null mutations but rather amino acid changes in evolutionarily-conserved residues. The tissue- specific defects these changes cause are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. To address these gaps in knowledge and provide insight into how mutations in EXOSC2 and EXOSC3 cause disease, we will develop a Drosophila model of RNA exosome-linked disease. EXOSC2 and EXOSC3 are evolutionarily conserved subunits, termed Rrp4 and Rrp40, respectively, in Drosophila. Using this model, we will exploit the Drosophila system to examine the role of the RNA exosome in vivo. The long-term goal of this project is to provide insight into how defects in subunits of a ubiquitous and critical RNA processing complex cause tissue- specific consequences. We will test the hypothesis that RNA exosome cap subunits mediate critical yet distinct activities of the RNA exosome in post-transcriptional regulation of gene expression in vivo. This hypothesis will be tested though three complementary aims: 1) Assess tissue-specific requirements for the Drosophila RNA exosome cap subunits Rrp4 (EXOSC2) and Rrp40 (EXOSC3); 2) Examine the functional consequences of amino acid substitutions in Rrp4 and Rrp40 that equate to disease-causing changes in EXOSC2 and EXOSC3, respectively; 3) Explore the potential mechanism by which amino acid changes in RNA exosome subunits cause distinct phenotypic consequences. Successful completion of these aims will further our knowledge of RNA exosome-linked disease and also provide invaluable insights into distinct tissue- specific consequences due to altered subunits within a single complex.